The present invention relates to a process for isolating and identifying a novel imidazo [4,5b] pyridinium molecule, referred to by the inventors as "pentosidine" from the extracellular matrix of humans and other mammals. The recently isolated imidazo [4,5b] pyridinium molecule, or pentosidine, is believed to be produced according to the non-enzymatic reaction of sugars with various amino acid or protein residues during the aging and/or degradation of proteins. In this regard, the pentosidine the molecule has been structurally characterized to consist essentially of a lysine and an arginine residue crosslinked by a pentose. Furthermore, the novel imidazo [4,5b] pyridinium or pentosidine molecule of the invention has been chemically synthesized in order to confirm the structural arrangement of the isolated molecule. The present invention is further directed to the use of the recently isolated, characterized, and chemically synthesized pentosidine molecule in various processes and/or compositions for studying the aging and/or degradation of proteins in humans and other mammals.
The extracellular matrix of humans and other mammals undergoes progressive changes during aging that are characterized by decreased solubility (Schnider, S. L., and Kohn, R. R., J. Clin. Invest. 67, pp. 1630-1635, 1981), decreased proteolytic digestibility (Hamlin, C. R., Luschin, J. H., and Kohn, R. R., Exp. Gerontol. 13, pp. 415-523, 1978), increased heat denaturation time (Snowden, J. M., Eyre, D. R., and Swann, D. H., Biochem. Biophys. Acta, 706, pp. 153-157, 1982) and accumulation of yellow and fluorescent material (LaBella, F. S., and Paul, G., J. Gerontol., 20, pp. 54-59, 1964). These changes, which affect particularly collagen-rich tissues and appear to be accelerated in diabetes, are thought to result from the formation of age-related intermolecular crosslinks.
Elucidation of the structure of these age-related intermolecular crosslinks has been for many years of major interest to gerontologists and collagen chemists for two principal reasons. First, there appears to exist an inverse relationship between mammalian longevity and aging rate of collagen (Kohn, R. R. in Testing the Theories of Aging (Adelman, R. C., and Roth, G. S., eds.) pp. 221-231, CRC Press, Inc., Boca Raton, Fla.) suggesting that the process which governs longevity may express itself at least partially in the aging rate of collagen. Second, the progressive increase in stiffness of collagen-rich tissues like arteries, lungs, joints and the extracellular matrix has been associated with age-related diseases such as hypertension, emphysema, decreased joint mobility and ability to fight infections. Thus, elucidation of the nature of extracellular matrix crosslinking in aging is of both practical and theoretical interest.
Along these lines, the present inventors and others previously postulated that the advanced Maillard or nonenzymatic glycosylation reaction which occurs between reducing sugars, e.g., glucose, and amino groups on proteins could explain some of the age and diabetes-related changes that affect long-lived proteins through browning and crosslinking (Monnier, V. M., and Cerami, A., Science, 211, pp. 491-493, 1981). However, direct demonstration of this hypothesis has not been possible since the structures of Maillard protein adducts and crosslinks were previously unknown.
In this regard, Cerami, et al., U.S. Pat. Nos. 4,665,192 and 4,758,583 reported the discovery of a new and useful fluorescent chromophore -2-(2-furoyl)-4(5)-2(furanyl)-1H-imidazole (FFI) and a method of utilizing this chromophore for inhibiting protein aging. However, the present inventors have demonstrated that the FFI compound described in these patents is merely an artifact of acid hydrolysis and alkalization with ammonia and is not one of the end products of extended non-enzymatic polypeptide glycosylation (Njoroge, et al., J. Biol. Chem., 263: 10646-10652, 1988).
However, notwithstanding the above, recent observations continue to suggest that some of the changes occurring in the aging process of collagen could be explained by the Maillard or nonenzymatic browning reaction which occurs in stored or heated foodstuffs (Monnier, V. M. and Cerami, A., Am. Chem. Soc. 215, 431, 1983). In this regard, reducing sugars react non-enzymatically with the free amino groups of the proteins to form insoluble, highly crosslinked, yellow and fluorescent products. Studies on the potential occurrence of the non-enzymatic browning reaction in vivo demonstrated an age-related increase in dura and skin collagen-linked fluorescence at 440 nm (excitation at 370 nm) and chromophores absorbing above 300 nm (Monnier, V. M., Kohn, R. R., and Cerami, A., Proc. Natl. Acad. Sci. 81, 583, 1984) (Monnier, V. M., Vishwanath, V., Frank, K. E., Elmets, C. A., Dauchot, P., and Kohn, R. R., New Engl. J. Med. 314, 403, 1986). Similar spectroscopical changes could be duplicated by incubating collagen with reducing sugars such as glucose, glucose-6-phosphate or ribose (Monnier, V. M., Kohn, R. R., and Cerami, A., Proc. Natl. Acad. Sci. 81, 583, 1984) (Kohn, R. R., Cerami, A., Monnier, V. M., Diabetes 33, 57, 1984). In addition, it was demonstrated that collagen incubated with these sugars was highly crosslinked suggesting that the sugar-derived fluorophores-chromophores could act as intra- or intermolecular crosslinks (Monnier, V. M., Kohn, R. R., and Cerami, A., Proc. Natl. Acad. Sci. 81, 583, 1984) (Kohn, R. R., Cerami, A., Monnier, V. M., Diabetes 33, 57, 1984).
The potential role of the Maillard reaction in these changes was further substantiated by the observation that non-enzymatic glycosylation which initiates the Maillard reaction was increased in diabetic and aging collagen and by observations in subject with Type I (insulin-dependent) diabetes that revealed a dramatic increase in collagen-linked fluorescence (Monnier, V. M., Vishwanath, V., Frank, K. E., Elmets, C. A., Dauchot, P., and Kohn, R. R., New Engl. J. Med. 314, 403, 1986) (Vishwanath, V., Frank, K. E., Elmets, C. A., Dauchot, P. J., Monnier, V. M., Diabetes 35, 916, 1986).
Although age-related acceleration of collagen browning may be explained by the Maillard reaction, the evidence presented for support of this hypothesis has been very circumstantial. More particularly, such evidence is based on spectroscopical changes of collagen with aging and diabetes in vivo with conspicuous similarities produced by the incubation of young collagen with reducing sugars in vitro. Because of uncertainty in the exact nature of the fluorescence produced during the aging of proteins, as well as the particular nature of the protein adducts and crosslinks involved therein, the present inventors initiated a study that resulted in the present invention with the ultimate aim of elucidating the nature of the collagen-linked fluorescence which increases in aging and diabetes.
In this regard, the present inventors conducted a systematic investigation of the chemical nature of the fluorescence that accumulates in aging human collagen. Two novel fluorophores, nicknamed "P" and "M", with excitation/emission maxima at 335/385 nm and 360/460 nm, respectively, were isolated from insoluble collagen following proteolytic digestion and chromatography (Sell, D. R., and Monnier, V. M., Conn. Tiss. Res. 19, pp. 77-92, 1989). An age-related effect was noted for both types of fluorophores (i.e. the presence of the fluorophores increased with age). Although fluorophore M was borohydride reducible and unstable to acid hydrolysis, thereby suggesting that M had an iminopropene-type configuration which substantiated, but did not prove, that glucose was responsible for its origin, the fluorescence properties of the 335/385 fluorophore, i.e. fluorophore "P", were found unchanged following acid hydrolysis in 6 N HCl for 24 hours at 110.degree. C. As a result of its resistance to acid hydrolysis, a larger quantity of fluorophore P was purified from acid hydrolyzed dura mater collagen and its structure was elucidated using .sup.1 H-NMR, .sup. 13 C-NMR and MS/MS fast atom bombardment spectroscopy. Structure elucidation of fluorophore "P" led to the discovery of a pentose-mediated protein crosslink named "pentosidine".